An Enzyme Transfers The Intact Oligosaccharides - Endo-M -

Endo-M is one of the enzymes known as endo-β-N-acetyl-glucosaminidases (endo-β-GlcNAc-ases). This enzyme was found by Yamamoto et al.¹⁾, in the culture fluid of Mucor hiemalis isolated from soil. Endo-M hydrolyzes N,N'-diacetylchitobiose moiety in oligosaccharides bound to the asparaginyl residue of various glycoproteins through N-glycosidic linkage. The efficacy of this enzyme comes from the fact that one N-acetylglucosamine residue remains bound to the protein while cleaving the N,N'-diacetylchitobiose moiety. The enzyme is thus able to transfer the intact oligosaccharide to suitable acceptors (Fig.1).

Yamamoto et al.²⁾ incubated an asialotransferrin glycopeptide with Endo-M in the presence of GlcNAc, then pyridylaminating (PA) oligosaccharides in the supernatant. In this experiment, they observed by HPLC that two separate PA-oligosaccharides had formed. One was the oligosaccharide released by hydrolysis, and the other was the released oligosaccharide that was transfered to GlcNAc. As acceptors, diacetylchiobiose and dansyl-asparaginyl N-acetylglucosamine (DNS-Asn(GlcNAc)) were also found to be effective. The enzyme was also capable of transferring high-mannose oligosaccharide to the acceptor diacetylchitobiose.

Haneda et al.³⁾ have transferred oligosaccharides with 9-fluorenylmethoxycarbonyl-asparaginyl-N-acetylglucosaminide (Fmoc-Asn(GlcNAc)) by incubating sialotransferrin glycopeptide, asialotransferrin glycopeptide and Man₆GlcNAc₂-Asn-peptide with Endo-M. Furthermore, synthetic hCG (β12-16)-GlcNAc-peptide has been subjected to transglycosylate with a sialo complex type oligosaccharide. An alternative synthetic method of peptide containing GlcNAc has been developed by Inazu et al.⁴⁾ This method uses Fmoc-Asn(GlcNAc), which was synthesized from aspartic acid containing N-terminal group protected by Fmoc group, and azide of GlcNAc instead of Fmoc-Asn-OH, and it applies a mixed acid anhydride method using dimethylthiophosphic acid (Mpt-MA) which generally shows poor responses toward hydroxyl group. By combining this method with Endo-M, many glycopeptides can be designed and easily prepared. Yamamoto⁵⁾ has compiled the outline of this methodology as the Chemo-Enzymatic Synthesis in his review. Endo-M can also be used to create new functions, by introducing glyco-chains, to the substances that originally do not have the specific functions. For example, Matsuda et al.⁶⁾ have introduced oligosaccharides to β-cyclodextrin. As the cyclodextrin is known to possess inclusion ability, it is expected that the product may become efficient drug carriers toward drug delivery system (DDS) by including and retaining a drug molecule within the cavity. By introducing oligosaccharides to cyclodextrin, the adducts can possess both the inclusion ability and lectin-recognition ability of oligosaccharides.

Fmoc-Asn(GlcNAc) was introduced to an 6-monoamino-β-cyclodextrin by using Mpt-MA method, along with the utilization of Endo-M, and thus β-cyclodextrin having N-linked oligosaccharide has been obtained. It was observed that the β-cyclodextrin carrying oligosaccharide had the high binding ability of lectin for concanavalin A (ConA).

Kojima, et al.⁷⁾ prepared a ruthenium complex with an α-glucosylated bipyridine. By using Endo-M, this group has obtained a novel glycocluster, which was formed by the introduction of a disialo complex-type oligosaccharide to the 4-OH group of a glucoside unit. The glycocluster carrying disialo complex-type oligosaccharide exhibited strong luminescence as well as excellent affinity toward type-A influenza viruses, and their luminescence intensity was markedly depressed by virus-binding. Therefore, their application to a luminescent probe to detect influenza viruses is expected.

As described above, Endo-M hydrolyzes the N,N'-diacetylchitobiose moiety in oligosaccharides bound to the asparaginyl residue of various glycoproteins. This hydrolysis can release oligosaccharides without causing any damage in the molecule, enabling the transfer of the released oligosaccharides to suitable acceptors. Unlike the conventional endo-β-GlcNAc-ase, it has been found that Endo-M is an enzyme with a broad substrate specificity, cleaving not only the high-mannose type and hybrid type of asparagine-linked oligosaccharides but also the complex type oligosaccharides in glycoproteins. In particular, the transglycosylation reaction of sialo complex type oligosaccharides was possible only by using Endo-M. Therefore, this methodology is expected to be applied to various fields, entailing a great demand of Endo-M among the researchers for future studies.

Yamamoto et al.⁸⁾ have cloned Endo-M genes, and this group has succeeded in obtaining transformant by introducing the recombinant vector to yeast.

Tokyo Kasei Kogyo Co., Ltd. successfully achieved largescale production of End-M by using the yeast. Please apply our product in the studies of oligosaccharides.